BHK-21-SC cell strain adapted to serum-free suspension culture and method for preparing vaccine antigen with cell strain

A BHK-21-SC, suspension culture technology, applied in veterinary vaccines, biochemical equipment and methods, medical preparations containing active ingredients, etc., can solve the problem of not being able to obtain high-titer virus solutions that meet the industrialization requirements of enterprises , to achieve the effect of promoting quality improvement

Inactive Publication Date: 2019-08-06
BEIJING VBIOSCI INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still no domestic enterprise to realize the application of suspension BHK-21 cells in chicken Newcastle disease vaccine. The reason may be that the existing technology can not obtain high-titer virus liquid that meets the industrialization requirements of enterprises by using suspension culture BHK-21 cells

Method used

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  • BHK-21-SC cell strain adapted to serum-free suspension culture and method for preparing vaccine antigen with cell strain
  • BHK-21-SC cell strain adapted to serum-free suspension culture and method for preparing vaccine antigen with cell strain
  • BHK-21-SC cell strain adapted to serum-free suspension culture and method for preparing vaccine antigen with cell strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1 Acclimatization of serum-free full suspension culture BHK-21 cells

[0071] 1 material

[0072] 1.1 Cell line: BHK-21 cells, purchased from Peking Union Medical College Cell Resource Center, are adherent cells for domestication.

[0073] 1.2 Reagents: DMEM medium, fetal bovine serum (FBS) and trypsin-EDTA were purchased from Gibco Company; newborn bovine serum (NBS) was purchased from Wuhan Sanli Company; TS-BHK105 serum-free cell culture medium (referred to as BHK105 medium ) comes from Yishengke (Shenzhen) Co., Ltd.

[0074] 1.3 Consumables: T75 square flask, 96-well cell culture plate, 6-well cell culture plate, 125ml shake flask, and 500ml shake flask were purchased from Corning Company.

[0075] 1.4 Instruments: shaker (model OS-200, Hangzhou Aosheng), 5L bioreactor (Sartorius), ordinary centrifuge, inverted microscope, constant temperature cell incubator, biological safety cabinet, automatic cell counter (model IC100) .

[0076] 2 methods

[0077] ...

Embodiment 2

[0119] Embodiment two: the screening of the highly adaptable BHK-21 cell line of Newcastle disease virus

[0120] 1 Materials and methods

[0121] 1.1 Materials

[0122] 1.1.1 Cell BHK-21-S cell line was domesticated and preserved by Beijing Dingchi Biotechnology Co., Ltd.;

[0123] 1.1.2 Virus Newcastle disease virus La Sota strain was identified and preserved by Qingdao Weilan Biological Products Co., Ltd.;

[0124] 1.1.3 Culture medium TS-BHK105 (abbreviated as BHK105 medium) and TS-BHK106 virus breeding enhancement solution (abbreviated as BHK106 virus breeding enhancement solution) are from Yishengke (Shenzhen) Co., Ltd., and DMEM medium is purchased from GIBCO company;

[0125] 1.1.4 Serum Fetal bovine serum was purchased from GIBCO;

[0126] 1.1.5 TPCK trypsin was purchased from SIGMA;

[0127] 1.1.6 1% chicken erythrocyte suspension, made by Beijing Dingchi Biotechnology Co., Ltd.

[0128] 1.2 Method

[0129] 1.2.1 Preparation of BHK-21-S monoclonal cell line in ...

Embodiment 3

[0150] Embodiment three: the domestication method of Newcastle disease virus vaccine strain La Sota strain cell adaptation virus

[0151] 1 Materials and methods

[0152] 1.1 Materials

[0153] 1.1.1 Newcastle disease La Sota strain chicken embryo seed virus (basic seed bank generation), HA titer is 1:512, cultivated, purified and identified by Qingdao Weilan Biological Products Co., Ltd.

[0154] 1.1.2 Experimental animals SPF chicken embryo and SPF chicken: purchased from Beijing Meria Weitong Experimental Animal Technology Co., Ltd.

[0155] 1.1.3 Antigens and antibodies for diagnosis

[0156] Avian influenza virus HI antigen was purchased from Harbin Veterinary Research Institute; AIV positive serum was purchased from Harbin Veterinary Research Institute; chicken NDV positive serum, ILTV positive serum, IBV positive serum, IBDV positive serum, EDSV positive serum and chickens without the above specific pathogens Negative serum was purchased from China Veterinary Drug Ad...

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Abstract

The invention discloses a BHK-21-SC cell strain adapted to serum-free suspension culture and a method for preparing a vaccine antigen with the cell strain. The BHK-21-SC cell strain is a juvenile golden hamster kidney cell strain, and has the preservation number of CGMCC No.16817. The cell strain is cultured according to the initial cell density of 0.5-0.6*10<6> cell / ml. 72 hours later, the cellsgrow to the cell density of 8-10*10<6> cell / ml. The screened and domesticated BHK-21 cell strain adapted to serum-free full suspension culture is used for production of a high-titer Newcastle diseasevirus solution, and a domestication process for preparation of Newcastle disease virus by the BHK-21 cell strain adapted to serum-free suspension culture is established.

Description

technical field [0001] The invention relates to the application of serum-free full suspension culture BHK-21 cells in the production of Newcastle disease vaccine, belonging to the field of veterinary biopharmaceuticals. Background technique [0002] At present, domestic Newcastle disease vaccines mainly use low-virulence live vaccines and oil adjuvant inactivated vaccines, which are routine prevention and control measures for poultry industry to prevent Newcastle disease. At present, the production of domestic Newcastle disease vaccine still mainly relies on the traditional "chicken embryo method" process to realize. When this method is produced on a large scale, because SPF chicken embryos (no specific pathogens, production of live vaccines) and common chicken embryos (production of inactivated vaccines) have shortcomings such as insufficient number of chicken embryos, high cost, and long production cycle of chicken embryos, domestic Not all vaccines are made from SPF chic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00A61K39/17A61P31/14
CPCA61K39/12A61K2039/5252A61K2039/552A61P31/14C12N5/0686C12N7/00C12N2760/18111C12N2760/18134C12N2760/18151
Inventor 李燕刘少奇张乾顺张凌云陈小娟孔俊飞李磊
Owner BEIJING VBIOSCI INC
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